Small-molecule control of super-Mendelian inheritance in gene drives

Amo, Víctor López del; Leger, Brittany S.; Cox, Kurt J.; Gill, Shubhroz; Bishop, Alena L.; Scanlon, Garrett D.; Walker, James A.; Choudhary, Amit

doi:10.1101/665620

Cited by 9 publications

(11 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Two cases are informative in this regard. First, e-CHACR-e did not copy when combined with Cas9, although in a split-drive configuration, without the Cas9-targeting gRNA, it exhibited modest drive ($60%) (Ló pez Del Amo et al, 2019). When combined with a cleavage-resistant Cas9* form, however, e-CHACR-e drove at levels similar to those in the split-drive experiments.…”

Section: Drive Performance Of Drive and Neutralization Systemsmentioning

confidence: 70%

Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives

Bulger

Gantz

et al. 2020

Molecular Cell

Self Cite

View full text Add to dashboard Cite

show abstract

Section: Drive Performance Of Drive and Neutralization Systemsmentioning

confidence: 70%

Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives

Bulger

Gantz

et al. 2020

Molecular Cell

Self Cite

View full text Add to dashboard Cite

show abstract

“…Briefly, we fused Escherichia coli dihydrofolate reductase (DHFR) domains to SpCas9 to promote its rapid proteasomal degradation in the absence of the stabilizing small molecule trimethoprim (TMP) 29,30 . We showed that TMP addition to the fruit fly diet stabilized our modified SpCas9 (DD2-Cas9) and allowed super-Mendelian inheritance control of a CopyCat active genetic element 28 .…”

Section: Gene Drive Process Reveals Predictable Resistant Allele Formmentioning

confidence: 98%

“…We recently developed a small-molecule-controlled system for use in active genetics approaches including CRISPR-based gene-drive systems 28 . Briefly, we fused Escherichia coli dihydrofolate reductase (DHFR) domains to SpCas9 to promote its rapid proteasomal degradation in the absence of the stabilizing small molecule trimethoprim (TMP) 29,30 .…”

Section: Gene Drive Process Reveals Predictable Resistant Allele Formmentioning

confidence: 99%

Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

Amo

Bishop

C.³

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

CRISPR-based gene drives spread through populations bypassing the dictates of Mendelian genetics, offering a population-engineering tool for tackling vector-borne diseases, managing crop pests, and helping island conservation efforts; unfortunately, current technologies raise safety concerns for unintended gene propagation. Herein, we address this by splitting the two drive components, Cas9 and gRNAs, into separate alleles to form a novel trans-complementing split-genedrive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This bi-component nature allows for individual transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a smallmolecule-controlled version to investigate the biology of component inheritance and use our system to study the maternal effects on CRISPR inheritance, impaired homology on efficiency, and resistant allele formation. Lastly, mathematical modeling of tGD spread in a population shows potential advantages for improving current gene-drive technologies for field population modification. Figure 1 -The trans-complementing gene-drive system (tGD) features simultaneous super-Mendelian inheritance of two transgenes.(A) Schematic of the tGD genetic arrangement with two elements that can be kept separated as different transgenic lines. The Cas9 transgene is inserted in the genomic location targeted by the gRNA-A, while a separate cassette expressing a tandem gRNA construct (gRNA-A, gRNA-B) is inserted at the location targeted by gRNA-B. Upon genetic cross, each gRNA combines with Cas9 to generate a double-strand DNA break at each locus on the wild-type allele. Each break is then repaired by homology-directed repair (HDR) pathway using the intact chromosome carrying the transgene as a template. (B) Outline of the genetic cross used to demonstrate tGD in fruit flies. F0 males carrying a DsRed-marked Cas9 transgene inserted in the yellow locus were crossed to females carrying a GFP-marked cassette containing two gRNAs (y1-e1) inserted in the ebony coding sequence. Trans-heterozygous F1 females (carrying both Cas9 and gRNAs) were crossed to wild-type males to assess germline transmission rates of the fluorophores marking the transgenes in the F2 progeny. The conversion event is indicated by the red and green triangles in the F1 females (C) Single F1 female germline inheritance output measured as GFP and DsRed marker presence in the F2 progeny. The black bar represents the inheritance average. The blue shading represents the deviation from the expected 50% "Mendelian" inheritance. Inheritance average, standard deviation, number of samples (n) and total number of flies scored in each experiment are represented over the graph in line with the respective data.

show abstract

“…Since we observed improved activity when using scaffold variants in both Culex quinquefasciatus and Drosophila melanogaster , we wondered whether these variants could be effectively used in a gene-drive strategy, and whether they would improve efficiency in these genetic systems. To test these hypotheses, we employed a split-gene-drive strategy (aka CopyCat) in Drosophila 19,42,44,45 . Briefly, a CopyCat element consists of a transgene containing a gRNA element (and a GFP marker) flanked by two homology arms, which match the targeted locus sequence on each side of a cut site generated by the gRNA within the transgene ( Fig.…”

Section: Grna Scaffold Variants Improve Gene Drive Efficiency In Drosmentioning

confidence: 99%

“…To compare transgenesis efficiency at the white gene in Drosophila using the "Loop" modification versus the original construct ( Fig. 4 ), each construct was separately injected into a Vasa -Cas9 line generated in our laboratory 42 (same line used for gene drive experiments). For these experiments, we waited until the injected G0 embryos became adults, then single-pair crossed them to white-eye mutant individuals.…”

Section: Drosophila Transgenesis Experimentsmentioning

confidence: 99%

Optimized CRISPR tools and site-directed transgenesis inCulex quinquefasciatusmosquitoes for gene drive development

Feng

Amo

Mameli

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we developed a Culex-specific Cas9/gRNA expression toolkit and used site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We showed that gRNA scaffold variants improve transgenesis efficiency in both Culex and Drosophila and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.

show abstract

Small-molecule control of super-Mendelian inheritance in gene drives

Cited by 9 publications

References 28 publications

Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives

Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives

Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

Optimized CRISPR tools and site-directed transgenesis inCulex quinquefasciatusmosquitoes for gene drive development

Contact Info

Product

Resources

About